PROJECT SUMMARY: Major human bacterial pathogens are often characterized by epidemics in which there is spread of closely related strains or clones. Epidemics are a hallmark of the major human pathogen group A Streptococcus (GAS), and much of what is known about bacterial epidemics is derived from GAS investigations. GAS is divided into M serotypes based on variation in the emm gene which encodes for the cell-surface, anti-phagocytic M protein that is the major GAS antigen impacting human immunity. Presently, GAS epidemics are thought to arise due to non- M protein factors, such as the proliferation of bacterial clones with increased virulence due to acquisition of new virulence factor encoding genes or alterations in virulence factor regulation. We have identified that serotype M4 strains that have recently been increasing in prevalence contain a novel form of M protein due a fusion between emm and its neighbor enn gene which differentiates these strains from ?pre-epidemic? M4 strains that contain both emm and enn. Genomic level comparison of a ?pre-epidemic? and an ?epidemic? M4 strain did not identify differences in virulence characteristics other than the emm/enn gene fusion These findings form the basis of this proposal which seeks to test the hypothesis that immune escape rather than a difference in virulence is responsible for the proliferation of ?epidemic? M4 strains. In specific aim 1, we will access 760 invasive M4 strains collected nationwide over the past 20 years by the Centers for Disease Control and Prevention to fully characterize the temporal and geographical spread of the ?epidemic? M4 strains. Using a combination of state- of-the-art whole genome sequencing approaches, we will define the molecular epidemiology of the M4 ?epidemic? strains and determine the range of genetic differences that separate ?epidemic? from ?pre-epidemic? isolates. In specific aim 2, we will leverage the data from specific aim 1 to choose representative isolates for a comprehensive virulence comparison in order to test the hypothesis that there is no significant difference in virulence between the ?epidemic? and ?pre-epidemic? isolates. Moreover, we will also test the hypothesis that ?epidemic? strains have undergone immune-escape from ?pre-epidemic? strains using an established GAS vaccination approach followed by strain challenge in a mouse model. The functional impact of vaccination against the emm/enn fusion encoding protein will be determined using enzyme-linked immunoabsorbance assays in the mouse model. Completion of these studies will challenge the prevailing dogma that increased virulence drives GAS epidemics and will have a potentially significant impact on GAS control strategies given that M protein is considered a prime vaccine candidate. Moreover, these studies have been designed to generate key preliminary data needed for more in-depth investigations of the molecular underpinning of bacterial epidemics, a critical aspect of bacterial pathogenesis.